MK is a growth factor discovered as the retinoic acid responsive gene product, and is a polypeptide that is rich in basic amino acids and cysteine and that has the molecular weight 13 kDa (Kadomatsu, K. et al.: Biochem. Biophys. Res. Commun., 151: 1312-1318, 1988; Tomomura, M. et al.: J. Biol. Chem., 265: 10765-10770, 1990). MK has 45% sequence homology to another heparin-binding protein referred to as pleiotrophin (PTN) or heparin-binding growth associated molecule (HB-GAM).
MK and PTN have common activities such as the neurotrophic factor activity (Li, Y.-S. et al.: Science, 250: 1690-1694, 1990; Merenmies, J. & Rauvala, H.: J. Biol. Chem., 265: 16721-16924, 1990; Muramatus, H. et al.: Dev. Biol., 110: 284-296, 1985), the enhancement of fibrinolytic system (Kojima, S. et al.: J. Biol. Chem., 270: 9590-9596, 1995), proliferation of various cells, transformation of NIH3T3 cells (Kadomatus, K. et al.: Brit. J. Cancer, 75: 354-359, 1997; Yokota, C. et al.: J. Biochem., 123, 339-346, 1998), and angiogenesis.
Thus, these MK family proteins are expected to be useful as drugs, and the development of high level expression system of these proteins has been strongly demanded. Natural MK and PTN proteins are not glycosylated. Therefore, the high level expression of these proteins with no glycosylation by recombinant DNA techniques, would be extremely useful not only for production of the proteins as drugs but also for structural and functional analyses of the proteins. In this invention, an unglycosylated MK family protein is referred to as an intact MK family protein. Herein, an MK family protein means a protein comprising at least the amino acid sequence of a mature protein of MK, PTN, or their functionally equivalent mutants. In addition, unglycosylated MK and PV are herein referred to as intact MK and intact PTN, respectively.
As the expression system for an MK family protein, the methanol-metabolizing yeast (referred to as “methylotrophic yeast” hereafter) is thought to be appropriate. In general, from the viewpoint that yeast in a unicellular eucaryote, abundant in molecular biological information, safe for humans, and easily cultured, it has been used as the host cell in the production of useful proteins by recombinant DNA techniques. In the secretory expression system of yeast, in particular, since the expressed protein in released to the outside of cells, the continuous culturing can be carried out with the expectation of a great deal increase in the production. Furthermore, since the labor to disrupt cells is saved, protein purification becomes easy.
In view of these facts, the present inventors have attempted to establish a high level secretory expression system of MK proteins using methylotrophic yeast, Pichia pastoris, as the host. An expression system of foreign genes by Pichia pastoris has been developed and reported in the production of hepatitis type B vaccine and a high-level secretory expression of invertase (Cregg, J. M. et al.: Bio/Technology, 11: 905-910, 1993). However, in the case where the secretion signal unique to MK protein is used for its expression, the expression level thereof is low (30-50 mg/L), and, furthermore, a majority of MK proteins thus expressed are bound to sugars originating in yeast which are different from those attached in animal cells. That is, the content of intact MK protein is extremely low. Use of MK proteins having sugars derived from yeast as drugs poses problems of antigenicity. Therefore, it becomes necessary to isolate and purify intact MK protein from expression products. However, it is highly difficult to isolate and purify proteins that have a common amino acid sequence but differ only in the attached sugars.
To increase an expression level of intact MK protein, the present inventors produced a large number of expression host strains in which the copy number of the expression cassette was increased, and allowed them to express the protein. They obtained yeast strains that produce an about 2-fold higher expression level than that obtained in conventional strains in the fermentor culture. However, they failed to acquire strains that highly express intact MK protein, indicating that an increase in the copy number is not directly related to the expression of MK protein.